A robust and sensitive qPCR assay for the detection and quantification of Xf bacteria in planta was used to relate Xf populations to leaf-scorch symptoms during PD. The results show that the nature of the Xf population is patchy across whole leaves. Leaves can exhibit severe leaf-scorch symptoms in the absence of high concentrations of Xf, and there is no apparent relationship between the severity of leaf-scorch symptoms and Xf concentration. These results demonstrate that localized high concentrations of Xf are not necessary for the formation of leaf-scorch symptoms, and suggest the involvement of an elicited plant response in Xf pathogenesis.
Quantifying Xf in planta
Developing the means of quantifying Xf from plant specimens has been an object of intense study, and a variety of approaches have been developed including immunolocalization, bacterial culture, ELISA, transgenic fluorescent Xf, PCR, and qPCR, all with their individual caveats. qPCR was chosen for the present study because of its superior sensitivity, relative ease in processing many samples, and ability to be adapted to an automated high throughput format. Xf concentrations of 103 cells g–1 fresh weight were regularly detected in this study, a level of sensitivity approximately 100 times greater than that reported for ELISA (105 cells g–1 fresh weight) (Krivanek and Walker, 2005). This provides for the quantification of Xf from much smaller sample sizes (e.g. and the ability to produce a much more detailed look at the nature of Xf colonization. Furthermore, the assay is highly reproducible, illustrated by coefficients of variation (CVs) in the standard curve of 1% or less (Table 1) compared with 35% on average for ELISA (Krivanek and Walker, 2005). Several qPCR assays for Xf have been developed in different plant species (Oliveira et al., 2002; Schaad et al., 2002; Li et al., 2003; Osman and Rowhani, 2006), but the majority have only employed the technology for detection, not quantification. Baumgartner and Warren (2005) is the only published report that has used qPCR in order to quantify Xf in grapevine but in a different species, the PD-resistant Vitis californica. Therefore, this is the first application of a qPCR methodology to the PD-susceptible, domesticated grapevine.
The crux of this methodology is isolating DNA template that is free from compounds having inhibitory effects on the reaction, and obtaining DNA from grapevine that is free from such compounds is notoriously difficult (Minsavage et al., 1994; Buzkan et al., 2003). In the current study, inhibition was uncommon, but also unpredictable. Inhibition occurred in some, but not other, individual leaf punch samples within the same leaf. This result should be of particular interest because the inhibition could lead to false negatives. The only solution is to evaluate every sample independently with the addition of an internal standard, as was done in this study.
The nature of Xf colonization and false negatives
Studies examining Xf in grapevine via fluorescent microscopy (Newman et al., 2003), by assessing a large number of field samples (Krell et al., 2006), as well as the study described here, all conclude that Xf has a patchy distribution within grapevine. Spatially variable concentrations are to be expected if the movement is dependent upon an irregular distribution of xylem conduit lengths and inter-vessel pit connections (Chatelet et al., 2006). As with the erratic inhibition discussed above, this knowledge impacts the interpretation of previously published findings and informs future experimental design. For example, many previous studies assessing Xf movement relied on only one or a few locations in order to evaluate Xf movement. This sampling approach, when used for diagnosis or for screens for resistant genotypes, may be wrought with false negatives (Fig. S2 in Supplementary material available at JXB online). Future studies would do better to utilize sampling methodologies that examine either a larger part of the plant, or a larger number of sampling locations in order to increase the likelihood of detecting Xf when it is present.
A number of previous studies in artificially inoculated grapevines demonstrate that Xf populations increase over time (Hopkins, 1985b; Fry and Milholland, 1990; Hill and Purcell, 1995); results confirmed by the current study. In the earlier studies Xf concentrations were often expressed in CFU ml–1 of extraction buffer, making direct comparisons with the present results difficult, but Hill and Purcell (1995) found populations of 108–109 CFU g–1 fresh weight, concentrations much greater than those detected in the majority of the samples that were positive for Xf in this study. This apparent discrepancy is explained by the fact that Hill and Purcell (1995) sampled at the point of inoculation, which is shown here to harbour concentrations of Xf (107–109 cells g–1 fresh weight) that are identical to those described in that work and much higher than in non-inoculated leaves. In fact, the majority of early studies quantifying Xf in grapevine did so by sampling at the point of inoculation, which may partly explain the common belief that high Xf concentrations are necessary for pathogenesis. Fry and Milholland (1990) reported populations of 104–106 CFU cm–1 of petiole at sites distal to the point of inoculation. Equivalent populations were regularly found in 1.5-cm-diameter leaf punch samples (see Fig. 3F, J–N).
In field samples the concentrations of Xf detected in positive leaves, 104–106 cells g–1, were similar to those reported in the resistant alternative host Vitis californica by Baumgartner and Warren (2005). That both these Vitis species harbour similar concentrations of Xf illustrates that symptom expression must involve more than simply high concentrations of Xf. The concentrations of Xf in field vines were also similar to those detected in greenhouse experiments with needle-inoculated Chardonnay grapevines. As far as is known, this is the first study that has quantified Xf populations from naturally inoculated Vitis vinifera from the field.
Xf populations and leaf scorch
This is also the first study that correlates symptoms with the concentration of Xf in leaves of PD-infected plants. As discussed above, the prevailing hypothesis to explain the formation of symptoms during PD is that vascular occlusion, largely attributed to Xf bacteria and their putatively associated gums, results in localized water deficits. It is clear from this and other recent studies that leaf-scorch symptoms form in the absence of localized high concentrations of, and substantial plugging of xylem vessels by, Xf (Newman et al., 2003; Alves et al., 2004; Krell et al., 2006). Newman et al. (2003) showed that the percentage of colonization and occlusion by Xf was greater in symptomatic leaves, although overall complete occlusion of vessels was extremely rare (2–4%) even for symptomatic leaves. In other species, Alves et al. (2004) reported that leaves exhibiting severe symptoms contained a higher percentage of Xf-colonized (not necessarily occluded) vessels than those leaves exhibiting mild symptoms. Again, in these species, leaves exhibited symptoms with as little as 8–26% of vessels colonized (Alves et al., 2004). In addition, these authors mention that preliminary research did not demonstrate a relationship between absolute population and symptom severity. In view of the low concentrations of Xf, patchy distribution of Xf, and the low frequency of occluded vessels, it is unlikely that leaf-scorch symptoms result from the obstruction of water movement by the Xf bacteria themselves. These observations do not discount that at times localized water deficits could result from high concentrations of Xf, but certainly it appears that this cannot account for all of the symptoms observed during pathogenesis. These observations do recall the question of the mechanisms by which Xf brings about PD.
Taken together, the results listed below are difficult to reconcile without the involvement of a systemic plant response in pathogenesis:
- (i) the absence of a strong relationship between leaf-scorch symptoms and Xf concentrations in individual leaves;
- (ii) a clear increase in the Xf population over time and in more symptomatic plants;
- (iii) the presence of leaf-scorch symptoms in tissues and leaves that have undetectable amounts of Xf.
If the grapevine responds systemically, visible symptoms will worsen throughout the whole of the plant, irrespective of the distribution of Xf, and any coincidence between high Xf populations and leaf-scorch symptoms will break down as pathogenesis progresses. Goodwin et al. (1988a) were the first to propose that PD is in essence accelerated leaf senescence, a hypothesis that has attracted little attention. Although Goodwin et al. 1988a) attributed this senescence to water stress, several other factors, Xf-derived elicitors among them, could bring about the same result (reviewed in Guo and Gan, 2005). An earlier study by Hopkins (1985a) demonstrated that plant growth regulators known to stimulate natural leaf senescence, such as ethylene, accelerate and intensify leaf-scorch symptoms during Xf pathogenesis, and more recent work has provided evidence that PD symptoms may result from an ethylene-mediated plant response (Perez-Donoso et al., 2007). Gene expression studies suggest that ethylene-mediated plant responses are also involved in the wilt diseases discussed above (Lasserre et al., 1997; Dowd et al., 2004; Wang et al., 2004). This could account for leaves exhibiting leaf-scorch symptoms in the absence of Xf and even for these leaves to exhibit water deficit. The results of this study serve to draw attention to the need to understand better the impact of the plant's developmental context and defence responses in Xf pathogenesis. Serious questions remain as to how vascular pathogens may be manipulating the plant to make the xylem environment more hospitable.